Mass spectrometry (MS) with ionization by matrix-assisted laser desorption and ionization (MALDI) has become a useful tool for the analysis of large molecules, e.g., biopolymers, such as proteins, peptides, oligonucleotides, DNA, RNA, etc. It is well known that the sensitivity of the analysis and the speed of automation are highly dependent on preparation and purity of the sample on the MALDI plate. Many of the issues associated with sample preparation for MALDI mass spectrometry are summarized in Bruker's UK patent application (GB 2332273A). In particular, salts can dramatically affect the quality of the resulting mass spectra due to adduct formation.
One existing method for salt removal utilizes reversed-phase sorbents such as C18-silica or divinylbenzene-based polymers. For example, a mixture of analytes is passed through a packed bed containing such sorbents, and the analytes adsorb to the packing material, while the salts are not retained. The adsorbed analytes are subsequently eluted from the sorbent with an appropriate solvent. Several commercially available products are available for this application.
However, all of these products require several manipulation steps prior to application of the sample onto the MALDI plate. These additional steps may lead to significant sample loss and potential re-contamination of the sample. In addition, samples typically become diluted during the sample preparation step because of the relatively large elution volumes that are required. Because the MALDI laser focuses on only a small percentage of the total area of the applied sample, only a small fraction of the applied diluted sample will be vaporized and detected, resulting in decreased sensitivity.
An additional problem with spotting a sample onto a traditional MALDI plate pertains to the drying of the sample on the plate. As the sample dries, the target analyte randomly concentrates in localized regions. As a result, analysis time for each sample increases, as the laser may need to search until it finds the target analyte that is sufficient for analysis. For example, when a drop of sample and matrix solution that is placed onto a clean metal sample support plate dries on a metal surface, the sample spot consisting of small matrix crystals spreads over the formerly wet area. In general, the wetted area is not uniformly coated. In aqueous solutions, most of the small crystals of the matrix generally begin to grow at the margin of the wet area on the metal plate and continue to grow toward the center of the wet area. Thus the analyte molecules are irregularly distributed, and the center of the spot is frequently empty or covered with fine small crystals. These crystals are not typically sufficient for MALDI ionization due to the high concentration of salts. Furthermore, the MALDI ionization yield and mass resolution fluctuate in the sample spot from site to site. It is often a troublesome process to find a favorable location on the sample spot with good analyte ion yield and good mass resolution. Consequently, the development of high sample throughput automation of MALDI mass spectrometry analysis has been hindered.
British patent application GB 2332273A describes a MALDI plate, coated with a Teflon-like hydrophobic coating having hydrophilic patches (“anchors”), which utilizes surface property (hydrophilic or hydrophobic) modification on the plate. After sample droplets are deposited onto the anchors, the droplets shrink during solvent evaporation, thereby centering themselves onto the anchor positions. Thus, MS detection sensitivity increases 10 to 100 times as compared to the conventional dried sample droplet preparation method described above, because the analyte is concentrated in smaller spots. The sample spots can be arranged in a precise grid to facilitate rapid, automated MALDI-MS. Such coated plates (AnchorChip™) are marketed by Bruker Daltonics®. However, these plates are incapable of retaining analyte molecules during a wash step, used to remove salts that may interfere with the analysis of the analyte.
U.S. Pat. Nos. 6,020,208 and 6,124,137 describe a MALDI-MS plate design in which the presentation surface of the target spot is derivitized with affinity molecules which allow for specific or nonspecific capturing of analytes of interest on the presentation surface. The sensitivity as well as the efficiency of the analysis by MALDI-MS are reportedly improved over conventional methods. The molecules that are not used for analysis may be subjected to further processing and subsequently further analysis on the presentation surface.
Published PCT applications WO 98/59360, WO 98/59361, WO 98/59362, and their corresponding European patent applications, EP 00990256, EP 00990257, and EP 00990258 describe plates, and methods of retentate chromatography for retaining selected molecules on a variety of adsorbents and using a variety of selectivity conditions. Upon selection of the highest affinity conditions direct MALDI-MS is used to analyze the selected molecule.
U.S. Pat. No. 5,480,526 describes the removal of salts from samples analyzed by MALDI-MS by using capillary electrophoresis methods prior to the analysis.
U.S. Pat. No. 6,104,028 describes the improvement of the sensitivity and resolution of MALDI-MS by improvement of the matrices.
U.S. Pat. Nos. 5,260,571, 5,281,538, and 5,308,978 describe improvement in sensitivity and resolution of detection by applying the matrix as a layer to the target spot of the MALDI-MS sample plate prior to the application of the sample, to thereby provide, after drying, an intimate mixture of sample and matrix material. Similarly, U.S. Pat. No. 5,595,636 describes the use of a thin lacquer-like smooth matrix layer applied prior to the application of the sample to assist in the desorption of the analyte molecules.
U.S. Pat. Nos. 5,770,860, 5,770,272, and 5,705,813 describe improved devices for use in connection with mass spectrometric analysis. In particular, U.S. Pat. No. 5,770,272 describes a nebulizing sprayer that deposits a continuous, homogeneous layer of MALDI matrix material on MALDI targets. U.S. Pat. No. 5,705,813 describes an integrated liquid handling system for manipulation of a sample prior to mass analysis by MALDI-MS. U.S. Pat. No. 5,770,860 discloses a method for loading sample supports of a mass spectrometer using a multiple pipette unit to simultaneously transfer multiple samples from microtiter plates. Repeated loading, in this fashion, generates high-density samples on the sample plate.
Nevertheless, a need exists for technological advancement of the MALDI-MS sample design that is capable of desalting samples directly on the MALDI plate while retaining the analyte and matrix on an underivatized surface, such that the salts may be washed away by the use of an appropriate solvent, e.g., water, and thereby reduce the number of sample manipulation steps while improving the sensitivity and resolution of detection by MALDI-MS by MALDI-MS. The use of small spots of the sample would localize the sample into a specific area, removing the requirement of searching for the sample location.